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14 ARTICLES PUBLISHED IN JoVE

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Biology

Transplantation of Cells Directly into the Kidney of Adult Zebrafish
Cuong Q. Diep 1, Alan J. Davidson 1
1Center for Regenerative Medicine, Massachusetts General Hospital

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

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Medicine

The Measurement and Treatment of Suppression in Amblyopia
Joanna M. Black 1, Robert F. Hess 2, Jeremy R. Cooperstock 3, Long To 3, Benjamin Thompson 1
1Department of Optometry and Vision Science, University of Auckland, 2Department of Ophthalmology, McGill University , 3Centre for Intelligent Machines, McGill University

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

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Neuroscience

Paired Whole Cell Recordings in Organotypic Hippocampal Slices
Chantelle Fourie 1, Marianna Kiraly 2, Daniel V. Madison *2, Johanna M. Montgomery *1
1Department of Physiology and Centre for Brain Research, University of Auckland, 2Department of Molecular and Cellular Physiology, Stanford University

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

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Bioengineering

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology
Vijay Rajagopal 1,2,3, Gregory Bass 2,3, Shouryadipta Ghosh 1,2,3, Hilary Hunt 2,4, Cameron Walker 5, Eric Hanssen 6, Edmund Crampin 2,3,4,7,8, Christian Soeller 9
1Cell Structure and Mechanobiology Group, University of Melbourne, 2Systems Biology Laboratory, Melbourne School of Engineering, University of Melbourne, 3Department of Biomedical Engineering, University of Melbourne, 4School of Mathematics and Statistics, Faculty of Science, University of Melbourne, 5Department of Engineering Science, University of Auckland, 6Advanced Microscopy Facility, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 7ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of Melbourne, 8School of Medicine, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, 9Living Systems Institute, University of Exeter

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

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Education

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers
Andria Pelentritou 1, Levin Kuhlmann 1, John Cormack 2, Will Woods 3, Jamie Sleigh 4, David Liley 1
1Centre for Human Psychopharmacology, Swinburne University of Technology, 2Department of Anaesthesia and Pain Management, St. Vincent's Hospital Melbourne, 3Brain and Psychological Science Research Centre, Swinburne University of Technology, 4Department of Anaesthesiology, University of Auckland

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

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Behavior

Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children
Lucy K. Goodman 1, Nicola S. Anstice 1,2, Suzanne Stevens 3, Benjamin Thompson 1,4, Trecia A. Wouldes 3
1School of Optometry and Vision Science, The University of Auckland, 2Discipline of Optometry, University of Canberra, 3Department of Psychological Medicine, The University of Auckland, 4School of Optometry and Vision Science, University of Waterloo

This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings.

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Cancer Research

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts
Denver D. Britto 1, Christopher J. Hall 1, Jonathan W. Astin 1
1Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

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Immunology and Infection

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes
Tanja Linnerz 1, Manju Kanamala 2, Jonathan W. Astin 1, Nicola Dalbeth 3, Zimei Wu 2, Christopher J. Hall 1
1Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, 2School of Pharmacy, Faculty of Medical and Health Sciences, University of Auckland, 3School of Medicine, Faculty of Medical and Health Sciences, University of Auckland

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

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Neuroscience

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke
Marie-Claire Smith 1,2,3, Suzanne J. Ackerley 1,2, Emma J. Monigatti 3, Benjamin J. Scrivener 3, Cathy M. Stinear 1,2
1Department of Medicine, University of Auckland, 2Centre for Brain Research, University of Auckland, 3Allied Health, Auckland District Health Board

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

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Medicine

Characterization of a Novel Human Organotypic Retinal Culture Technique
Charisse Y. J. Kuo *1, Henry H. Louie 1, Ilva D. Rupenthal 1, Odunayo O. Mugisho *1
1Buchanan Ocular Therapeutics Unit, Department of Ophthalmology and the New Zealand National Eye Centre, University of Auckland

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment epithelium-choroid (RPE-choroid) and sclera.

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Developmental Biology

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells
Aneta Przepiorski 1, Amanda E. Crunk 1, Teresa M. Holm 2, Veronika Sander 2, Alan J. Davidson 2, Neil A. Hukriede 1,3
1Department of Developmental Biology, University of Pittsburgh, School of Medicine, 2Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 3Center for Critical Care Nephrology, University of Pittsburgh, School of Medicine

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

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Biology

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis
Golnoush Madani *1, Erwin Lamping *1, Hee Ji Lee 1, Masakazu Niimi 1,2, Alok K. Mitra 3, Richard D. Cannon 1
1Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, 2Department of Microbiology, Faculty of Medicine, Chulalongkorn University, 3School of Biological Sciences, University of Auckland

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

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Bioengineering

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo
Jarrah M. Dowrick 1, Alex J. Anderson 1, Ming L. Cheuk 1, Kenneth Tran 1, Poul M. F. Nielsen 1,2, June-Chiew Han 1, Andrew J. Taberner 1,2
1Auckland Bioengineering Institute, University of Auckland, 2Department of Engineering Science, University of Auckland

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

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Biology

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines
Akshata Anchan 1,2, James J.W. Hucklesby 2,3, E. Scott Graham 1,2, Catherine E. Angel 3
1Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 2Centre for Brain Research, University of Auckland, 3School of Biological Sciences, University of Auckland

Here we demonstrate the technique of using impedance-based biosensors: ECIS and cellZscope, for measuring brain endothelial barrier strength. We detail the preparation and technique of adding various stimuli to an in vitro model of the brain endothelium. We measure, record, and give a representative analysis of the findings.

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